Metoda Lipnik-Stangelj

Background/Objectives: Synthetic cannabinoid receptor agonists (SCRAs) are associated with severe neurotoxicity, but the cellular mechanisms underlying their effects remain poorly defined. We investigated the effects of Cumyl-PINACA (SGT-24), a carboxamide-type SCRA derived from cumylamine, on rat cortical neurons and astrocytes. Methods: Primary rat cortical neurons and astrocytes were exposed to 1–10,000 nM SGT-24. Metabolic activity, mitochondrial function, morphology, and cell death were evaluated. Selective antagonists of cannabinoid receptor type 1 (CB1), G protein-coupled receptor 55 (GPR55), peroxisome proliferator-activated receptor gamma (PPARγ), and transient receptor potential cation channel subfamily V member 1 (TRPV1) were used to probe the involvement of these receptor pathways. Results: SGT-24 decreased metabolic activity in both cell types in a concentration- and time-dependent manner, with greater potency in neurons (IC₅₀ = 13.2 nM) than in astrocytes (IC₅₀ = 39.8 nM). After 24 h, maximal effects were observed at 100 nM in neurons and 500 nM in astrocytes, reducing metabolic activity by 45.2% and 36.2%, respectively. At these concentrations, mitochondrial membrane potential decreased to 57.6% and 54.1% of control, while cellular ATP levels fell to 51.6% and 52.5%, respectively. Neurons predominantly exhibited early apoptosis (21.9% of cells vs. 3.1% in controls), whereas astrocytes showed mainly 7-aminoactinomycin D (7-AAD)-positive cell death (19.3% vs. 8.2% in controls). Pharmacological inhibition of CB1, TRPV1, and PPARγ attenuated SGT-24-induced metabolic impairment, mitochondrial dysfunction, and apoptosis in neurons, whereas inhibition of CB1 and PPARγ reduced astrocytic toxicity. Conclusions: SGT-24 exerts potent, cell-type-dependent neuroglial toxicity associated with mitochondrial dysfunction and distinct cell death patterns, suggesting the involvement of cannabinoid receptor-dependent and non-cannabinoid signalling mechanisms. The nanomolar potency of SGT-24 underscores the toxicological risk posed by high-potency SCRAs.

Cannabinoid receptors occupy strategic control nodes within motor circuitry, making them potential targets for modulating different motor manifestations. They are positioned both within basal ganglia circuits that regulate movement and within spinal circuits that control skeletal muscle tone. Consequently, cannabinoids have been studied across diverse motor disorders, most notably in movement disorders and tone disorders, particularly those resulting in spasticity. Because motor control spans multiple anatomically and functionally distinct levels, relating cannabinoid signaling to effects on motor function is not straightforward. Limited understanding of cannabinoid receptor distribution has led to cannabinoids being tested even in disorders where receptor localization would predict little or no benefit. Mapping receptor distribution within individual motor circuits and integrating them with their pharmacological effects can help anticipate how cannabinoid signaling shapes motor output. Combined with characteristic motor manifestations, one can identify motor disorders in which cannabinoids may have therapeutic value. In this review, we integrate existing evidence to place cannabinoid receptors within key motor pathways, ranging from basal ganglia circuits controlling movement to peripheral mechanisms governing muscle tone. We consider both cannabinoid 1 receptor (CB₁R) and cannabinoid 2 receptor (CB₂R), with CB₂R gaining attention only recently for its potential relevance within the central nervous system. Building on this framework, we infer how cannabinoids acting at these sites may modulate motor control, and consequently, influence motor manifestations across major motor disorders. Finally, we examine how these distribution-based expectations align with available clinical observations.

The influence of cannabidiol (CBD) on brain development is inadequately understood. Since CBD is considered a non-intoxicating drug, it has attracted great interest concerning its potential medical applicability, including in pregnant women and children. Here, we elucidated the response of perinatal rat cortical neurons and astrocytes to CBD at submicromolar (0.1, 0.5, 1, 5 µM) concentrations attainable in humans. The effect of CBD was concentration- and time-dependent and cell-specific. In neurons, 0.1 µM CBD induced an early and transient change in mitochondrial membrane potential (ΔΨm), ATP depletion, and caspase-8 activation, followed by rapid ATP recovery and progressive activation of caspase-9 and caspase-3/7, resulting in early apoptotic cell death with reduction and shortening of dendrites, cell shrinkage, and chromatin condensation. The decrease in neuronal viability, ATP depletion, and caspase activation due to CBD exposure was prevented by transient receptor potential vanilloid 1 (TRPV1) antagonist. In astrocytes, 0.5 µM CBD caused an immediate short-term dysregulation of ΔΨm, followed by ATP depletion with transient activation of caspase-8 and progressive activation of caspase-9 and caspase-3/7, leading to early apoptosis and subsequent necroptosis. In astrocytes, both TRPV1 and cannabinoid receptor 1 (CB₁) antagonists protected viability and prevented apoptosis. Given that CBD is a non-intoxicating drug, our results clearly show that this is not the case during critical periods of brain development when it can significantly interfere with the endogenous cannabinoid system.

Purpose: In this study, the effect of gabapentin on the regulated cell death of astrocytes in primary culture was examined. Because astrocytes are relatively resistant to decay by apoptotic pathways, the effect of different concentrations of gabapentin on apoptosis in necroptosis was tested as another form of regulated cell death. In addition, the impact of gabapentin on the death of astrocytes that were exposed to ethanol was also examined. Methods: Primary cultures of astrocytes that were obtained from the brain cortex of newborn rats were used as the experimental model. Cells were exposed to different concentrations of gabapentin only, ethanol only or to a combination of ethanol and gabapentin. Using flow cytometry, the proportions of viable, early apoptotic, necroptotic, and secondary dead cells were determined. Results: The effect of gabapentin on early astrocytic apoptosis and necroptosis was dependent on concentration. In concentrations of up to 10 μg/mL, gabapentin did not affect astrocyte deaths; whereas at higher concentrations, the proportion of necroptotic cells increased. The concomitant exposure of the cells to gabapentin (10 μg/mL) and ethanol (100 mM) for 24 hours did not significantly affect cell death caused by ethanol. For cells that are exposed to 50 mM ethanol for 7 days, gabapentin slightly reduced the proportion of necrotic cells. Conclusion: Gabapentin did not affect the viability of astrocytes in concentrations up to 10 μg/mL. The concomitant exposure of astrocytes to ethanol and gabapentin for 24 hours did not reduce the toxicity of ethanol. In astrocytes that are chronically exposed to ethanol, gabapentin slightly reduced the effect of ethanol on necroptosis.

The discovery of the endocannabinoid system has raised public interest in the medicinal use of cannabis, phytocannabinoids, and synthetic cannabinoids, which has always been closely regulated due to their psychotropic effects and potential abuse. The review takes a quick look at the current legal framework in the European Union, which regulates cannabis use and cultivation for medicinal purposes in line with the United Nations Conventions on the production, trade, and use of cannabis, phytocannabinoids, and synthetic cannabinoids. And while the EU legislation precisely defines requirements and marketing authorisation procedures for medicinal products for all EU member states, there is no common regulatory framework for magistral and officinal preparations containing cannabinoids, as they are exempt from marketing authorisation. Instead, their regulation is left to each member state, and it is quite uneven at this point, mainly due to cultural and historical differences between the countries, leading to different access to non-authorised medicinal products. Therefore, to meet great public interest, harmonised approaches on cannabinoid-containing products without marketing authorisation would be welcome to level the playing field in the EU.

Background: Astrocytes maintain central nerve system homeostasis and are relatively resistant to cell death. Dysfunction of cell death mechanisms may underlie glioblastoma genesis and resistance to cancer therapy; therefore more detailed understanding of astrocytic death modalities is needed in order to design effective therapy. The purpose of this study was to determine the effect of VAS2870, a pan-NADPH oxidase inhibitor, on staurosporine-induced cell death in astrocytes. Materials and methods: Cultured rat astrocytes were treated with staurosporine as activator of cell death. Cell viability, production of reactive oxygen species (ROS), and mitochondrial potential were examined using flow cytometric analysis, while chemiluminescence analysis was performed to assess caspase 3/7 activity and cellular ATP. Results: We show here for the first time, that VAS2870 is able to prevent staurosporine-induced cell death. Staurosporine exerts its toxic effect through increased generation of ROS, while VAS2870 reduces the level of ROS. Further, VAS2870 partially restores mitochondrial inner membrane potential and level of ATP in staurosporine treated cells. Conclusions: Staurosporine induces cell death in cultured rat astrocytes through oxidative stress. Generation of ROS, mitochondrial membrane potential and energy level are sensitive to VAS2870, which suggests NADPH oxidases as an important effector of cell death. Consequently, NADPH oxidases activation pathway could be an important target to modulate astrocytic death.

The involvement of serotonin (5-HT) in chronic pain mechanisms is established. 5-HT inhibits central painful stimuli, but recent data suggests that 5-HT could also enhance pain stimulus from the periphery, where mast cells play an important role. We aimed in our study to clarify the influence of selected tricyclic antidepressants (TCAs) on mast cell function: secretion, uptake, and reuptake of 5-HT, that could interfere with 5-HT levels and in this way contribute to the generation of pain. As an experimental model, we used isolated rat peritoneal mast cells and incubated them with selected TCAs (clomipramine, amitriptyline, doxepin, and imipramine) under different experimental conditions. 5-HT release, uptake, and reuptake were determined spectrofluorometrically. We showed that TCAs were able to inhibit 5-HT secretion from mast cells, as well as uptake of exogenous 5-HT and reuptake of secreted 5-HT back into mast cells. The effects of TCAs were concentration dependent; higher concentrations of TCAs inhibited the secretion of 5-HT induced by compound 48/80, whereas lower concentrations of TCAs inhibited 5-HT uptake. The most effective TCA was halogenated clomipramine. As TCAs are well introduced in chronic pain treatment, the insight into mechanisms of action is important for an understanding of their effect in various pain conditions.

Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec-1). Different forms of a cell death were detected using flow cytometry. We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10(-7) M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10(-6) M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis.